Controlled reversible poration for preservation of biological materials

ABSTRACT

A preservation method for biological material having cell membranes includes reversibly porating the cell membranes; loading a bio-protective agent having bio-preservation properties to a predetermined intracellular concentration; preparing the bio-protective agent loaded biological material for storage; storing the biological material; recovering the stored biological material from storage; and reversing the cell membrane poration. H5 α-toxin, a genetically engineered mutant of Staphylococcus aureus α-hemolysin, may be used as a porating agent. Non-permeating sugars such as trehalose and sucrose may be used as the bio-protective agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

Not Applicable.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was partially supported by NIH grants DK 46270 arid NS 26760.

FIELD OF THE INVENTION

The present invention relates to the preservation of biological tissueusing reversible controlled poration methods to load intracellularprotective agents to preserve cells by freezing and/or drying.

BACKGROUND OF THE INVENTION

With recent advances in cell transplantation, tissue engineering andgenetic technologies, the living cell is becoming an importanttherapeutic tool in clinical medical care. From the use of livingartificial skin and bone material to treat burn and trauma victims, tobioartificial devices and direct transplantation of cellular material totreat the increasingly long list of genetically-based diseases, livingcells are increasingly incorporated into comprehensive treatment. Insuch a construct, the exogenous cells perform the multitude of complextasks which the diseased tissue cannot. Successful long-termpreservation and storage of mammalian cells is critical to the successof this type of medical care. Current preservation technology, includingcryobiological technology, often requires rather complicated freezingand thawing protocols which may be specific for cell type, eachrequiring some variation of a cryopreservation agent (CPA) cocktail tohelp the cell overcome freezing stresses.

Most traditional cryopreservation protocols include the addition of1.0-2.0 M of penetrating CPAs such as DMSO, glycerol, and ethyleneglycol. Small carbohydrate sugars, such as trehalose, sucrose, andmaltose have physicochemical properties (e.g., glass formation) for useas CPAs which are superior to traditional CPAs, however, mammalian cellmembranes are not practically permeable to these materials.

In order to provide the preservation of mammalian cells necessary forapplication of living cells as a therapeutic tool in clinical medicalcare, new protocols for preserving living nucleated cells using lowlevels of non-toxic preservation agents and having simple proceduresapplicable to a variety of cells must be developed.

SUMMARY OF THE INVENTION

The present invention provides a method for preserving living cells thatbegins with the reversible poration of the cell membranes. In onepreferred embodiment, this reversible poration is accomplished using H5α-toxin, a genetically engineered mutant of Staphylococcus aureusα-toxin having five of its native residues replaced with histidines.Once porated, the biological material is loaded to a predeterminedintracellular concentration with a bio-preservation agent such as asugar having bio-preservation properties. The method of the inventionmay advantageously use low levels, less than or equal to about 1.0 M, ofintracellular sugar and may use intracellular sugar alone as theprotective agent, in combination with other intracellular sugars, or incombination with traditional penetrating CPAs.

The biological material is then prepared for storage. In general, thematerial may be prepared for storage by freezing and/or drying. In anexemplary embodiment, a simple plunge freezing technique is shown tohave very high yield in the method of the invention. A vacuum dryingprotocol is also shown to result in post storage viability. In addition,air drying is well as freeze drying techniques may be employed.

Once the biological material is prepared for storage, it is stored in amanner appropriate to its preparation. Frozen material can be stored atcryogenic temperatures and dried material can dry stored at ambient orother temperatures as appropriate. Recovery of stored material is alsoappropriate to the method of its preparation for storage. Dried materialcan be rehydrated and frozen materials can be thawed. Cell membraneporation reversal may also be accomplished during the recovery step.When using H5 α-toxin, poration reversal may be accomplished by additionof μM concentrations of Zn²⁺ ions. This may be accomplished by placingthe cells into a culture medium having a sufficient concentration ofsuch ions.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be more fully understood by reference to thefollowing detailed description when considered in conjunction with theaccompanying drawings, in which:

FIG. 1 is flow chart showing steps in the method of the invention;

FIGS. 2A-G illustrate functional testing of cells loaded with 0.4Mintracellular trehalose, plunge frozen in liquid nitrogen, and thawed at37° C.;

FIGS. 2A, C, E and G illustrate plating efficiency at 3 hours;

FIGS. 2B, D, F and G illustrate population survival and growth at 18hours;

FIG. 3 illustrates the effects of trehalose in the method of theinvention as an intracellular protective agent for 3T3 fibroblastsplunge-frozen in liquid nitrogen and thawed at 37° C.;

FIG. 4 shows the effects of trehalose concentration in the method of theinvention on the survival rate for 3T3 fibroblasts plunge-frozen inliquid nitrogen and thawed at 37° C.; and

FIG. 5 illustrates fibroblast viability after vacuum drying according tothe method of the invention estimated by counting attached cells withcharacteristic spread morphology and the ability to grow and divide.

DETAILED DESCRIPTION OF THE INVENTION

A method for preserving biological tissue of the invention, illustratedin FIG. 1, starts with the selection or isolation of the cells or tissueto be preserved 10. While the method of the invention may be used forthe preservation of any biological material having lipid membranes, itis most useful for the preservation of living nucleated cells and, inparticular, mammalian cells such as fibroblasts, hepatocytes,chondrocytes, keratinocytes, islets of Langerhans, granulocytes, andhematopoeitic cells including bone marrow cells, platelets, red bloodcells and others.

The target cells are then porated or permeabilized 20 to facilitate theloading of a bio-preservation solution. Preferably, the target cells arereversibly porated, that is, pores are opened in the cell membranes ofthe target cells, but the poration process is controllably reversible asdescribed herein. In one embodiment of the invention, the cell membranesare permeabilized by a genetically engineered mutant of Staphylococcusaureus α-toxin known as H5 (due to the replacement of five of its nativeresidues with histidines). The structure of H5 is described in Song etal., "Structure of Staphylococcal α-hemolysin, a heptamerictransmembrane pore," Scienice, 274, 1859-1866 (1996). H5, a 293 aminoacid, 34 kDa protein forms uniform 2-nm homoheptameric transmembranepores upon introduction into lipid bilayers. The amount of poration inthe cell membranes is dose-dependent and the uptake of sugars throughthese pores is rapid. [Russo et al., "Reversible permeabilization ofplasma membranes with an engineered switchable pore," Nature Biotech.15, 278-282 (1997).]

Due to a targeted mutation, H5 pores are uniquely capable of beingtoggled between an open and closed state by the removal or addition ofμM concentrations of Zn²⁺ ions, respectively. [Walker et al., "Apore-forming protein with a metal-actuated switch," Protein Eng. 7,655-662 (1994); Bayley, H., "Building doors into cells," Sci. Am. 277,62-67 (1997).] The activity of the switch allows permeabilized cells toregain their original permeability and, as a result, maintain theviability and ultra-structural integrity.

Other poration agents may also be used with the method of the inventionto reversibly porate target cells. Other variants of the Staphylococcusaureus α-toxin are available having different poration characteristics.In addition, under certain conditions it is possible to createreversible pores in cell membranes using wild type Staphylococcus aureusα-toxin (WT). WT may not be stable in all membranes when the cells aremetabolically active (e.g., in a culture medium). As a result, for cellsthat can remain metabolically active for a longer period of time thanthe WT pores remain stable, the cells can shed even WT pores to reversethe poration effect. While some cells are too sensitive to survive forthe required amount of time, other cells, such as 3T3 fibroblasts, mayremain active in a porated state for up to 30 minutes and could bereversibly porated using WT.

Typically an H5 pore is about 2 nm in diameter. This size allowsmolecules having a molecular weight of up to about 1000 to enter thetarget cells through the H5 pores and permits many otherwisenon-permeating bio-preservation agents to be used intracellularly. Otherself-assembling membrane toxins capable of opening larger pores can beused or modified for use with the invention. While the application oflarger pores may permit the use of larger molecules as intracellularbio-preservation agents, larger pores can have damaging effects on thetarget cell as well. For example, if the pores are large enough to admitthe porating agent itself into the target cell, the porating agent mayexcessively permeabilize internal membranes and potentially causeirreparable damage to the target cell.

The target cells may be prepared for poration by trypsinizing, washingand suspending in a HEPES(N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) BufferedSaline (HBS) solution. Preferably, all cell preparation should occur inHBS so that no antibodies against the poraling agents or proteolyticenzymes compromise the porating process. In order to porate the targetcells, the HBS suspending solution should also contain a porating agentsuch as H5. As in the examples described below, the ratio of poratingagent to target cell in the solution may be 2×10⁶ cells/ml of the targetcells to 25 μ/ml of porating agent. The cells should be incubated in theporating agent containing solution for 10 minutes to allow for properpore formation. [Walker et al., "An intermediate in the assembly of apore-forming protein trapped with a genetically engineered switch,"Chem. Biol. 2, 99-105 (1995).] Of course, the precise amounts and timesrecited herein may be varied by a person of ordinary skill in the art inkeeping with the invention where necessary to account for varyingcircumstances and desired effects. For example, some target cells mayhave receptors for H5 and may exhibit increased poration.

Following the permeabilization or poration step 20, a bio-preservationagent is loaded into the porated cell 30. The poration step of theinvention allows otherwise non-permeating bio-preservation agents to beused intracellularly. As used herein, the term "non-permeating" refersto a compound that does not permeate the cell membrane of a mammaliancell under normal conditions in the necessary amounts in a reasonableamount of time (minutes to hours). These bio-preservation agents caninclude sugars as well as other non-permeating compounds either alone,mixed together, or in solution with other traditional bio-preservationagents. It is also possible that new bio-preservation agents will besynthesized specifically for intracellular application through pores ofthe type described herein.

Sugars having a stabilizing or preserving effect on biological materialare especially useful in the present method. Exemplary sugars includetrehalose, glucose, sucrose and maltose. Trehalose, a non-reducingdisaccharide of glucose that is normally impermeant to mammalian cellmembranes, is the most preferred sugar for use with the present method.It has an exceptional ability to stabilize and preserve proteins,viruses, and bacteria as well as an unusual ability to form stableglasses at high temperatures. Trehalose has physicochemical propertiesfor use as a mammalian cell cryoprotective agent (CPA) that are farsuperior to traditional agents. Further, trehalose, contained in manyfood products, is relatively non-toxic and may allow forcryopreservation protocols which do not required CPA removal, resultingin an infusible end product. Sucrose, which has properties similar tothose of trehalose and which is widely available and relativelyinexpensive, may also be preferred for certain applications.

Sugar may be added to the cell suspension in an HBS solution to thefinal desired concentration. Porated cells may be incubated in the sugarcontaining solution for 45 minutes. A 45 minute interval has beendemonstrated to be sufficient for the uptake of sucrose and trehalosefor 3T3 fibroblast cells porated with 25 μ/ml H5. Of course the lengthof time required for sugar uptake may vary with the type of cell and thelevel of cell poration. Sugar uptake may be measured by both the uptakeof radiolableled sugar into porated cells as well as by the volumetricresponse of porated cells placed in a hypertonic sugar solution,yielding statistically correlative results.

Most traditional cryopreservation protocols include the addition of1.0-2.0 M of penetrating cryoprotectants (CPAs) such as DMSO, glycerol,and ethylene glycol. However, using the method of the invention, smallcarbohydrate sugars such as trehalose, sucrose and maltose, to whichmammalian cell membranes are not practically permeable, may be loaded toconcentrations less than or equal to about 1.0 M, preferably less thanor equal to about 0.4 M, and most preferably, the suspended, poratedcells are loaded with about 0.2 M sugar.

In addition, reversible poration can improve the loading ofconventional, permeating CPAs. Conventional CPAs used in conventionalpreservation procedures require tedious loading and removal steps.Typically, when a cell is exposed to a penetrating CPA such as DMSO, thecell initially shrinks because the permeability of the plasma membraneto water is significantly greater than its permeability to DMSO. Next,as the DMO slowly penetrates the membrane, the cell swells untilequilibrium is achieved and loading of the CPA is complete. If the CPAis fully loaded in one step (e.g., 1-2 M DMSO), the initial cellshrinkage is fast and excessive, resulting in "osmotic" damage and celldeath.

To prevent cell death, conventional CPAs are added in multiple steps.Typically, each step (depending, of course, on cell type) takes 15 to 45minutes to load approximately 0.5 M CPA. The overall load process canthus take from 30 minutes to 2 hours or more. Similar circumstancesoccur during removal of the CPA from the cells. This long exposure toCPAs such as DMSO is "toxic" and can cause cell lysis. Applyingconventional CPAs in the method of the invention using a reversibleporation step increases the permeability to penetrating CPAs andaccordingly lessens cell shrinkage and CPA loading times. As a result,the method of the invention can decrease both "osmotic" and "toxic"injuries, even using conventional CPAs.

It may also be beneficial to add certain high molecular weightbio-preservation agents that do not permeate through the pores. One suchagent is raffinose. Raffinose attracts water that may diffuse into thebiological material by forming a pentohydrate and stabilizes the glassystate against increases in moisture content (e.g. though cracked vials,etc.). Dextran of various molecular weights, having good glass formationproperties, may be used extracellularly to allow increases in thestorage temperature of a frozen stored sample. Other large moleculeswhich do not permeate through H5 or similar pores may also be usedextracellularly with the method of the invention to enhance the outcomeof a particular preservation protocol.

Following the bio-preservation agent loading step 30, the biologicalmaterial is prepared for storage 40. A variety of methods for freezingand/or drying may be employed to prepare the material for storage. Inparticular, three approaches are described herein: vacuum or air drying50, freeze drying 60, and freeze-thaw 70 protocols. Drying processeshave the advantage that the stabilized biological material may betransported and stored at ambient temperatures.

In contrast to current cryobiological technology which often requirescomplicated freezing and thawing protocols which may need to be specificto each type of cell being preserved, the method of the invention allowsfor a simple freeze-thaw protocol having a high survival rate, makingcold storage a good choice as well. In the simple freezing step 80 ofthe invention, approximately 1 ml of cell suspension (10⁶ cells/ml) maybe placed into a cryovial, clipped into a cryovial holder, and plungedinto liquid nitrogen (LN₂) for a sufficient time to bring the suspensionto a cryopreservation temperature.

The suspended material can then be stored 90, 100 at cryopreservationtemperatures, for example, by leaving the vials in LN₂, for the desiredamount of time. The suspended material can then be recovered fromstorage 110 by thawing 120 in a 37° C. water bath with continuous, mildagitation for 5 minutes. For later analysis by the methods describedbelow, thawed cells can diluted in Dulbecco's Modified Eagle Medium(DMEM) solution containing 10% bovine calf serum which has sufficientZn²⁺ to seal the H5 pores to reverse the cell poration 170 and promotecell recovery, or in any other appropriate dilution medium.

Protocols for vacuum or air drying 50 and for freeze drying 60 proteinsare well characterized in the art [Franks et al., "Materials Science andthe Production of Shelf-Stable Biologicals," BioPharm, October 1991, p.39; Shalaev et al., "Changes in the Physical State of Model Mixturesduring, Freezing and Drying: Impact on Product Quality," Cryobiol. 33,14-26 (1996).] and such protocols may be used to prepare cellsuspensions for storage with the method of the invention.

An evaporative vacuum drying protocol 130 useful with the method of theinvention for preserving 3T3 murine fibroblasts may include placing 20μl each into wells on 12 well plates and vacuum drying for 2 hours atambient temperature. Of course, other drying methods could be used,including drying the biological material in vials.

Biological material prepared in this manner may be stored dry 140, andrehydrated by diluting in DMEM which contains serum (and sufficient Zn²⁺to seal the H5 pores) to reverse the cell poration 170 and promote cellrecovery.

A method of the invention using freeze drying 60 to prepare thebiological material for storage 40 begins with freezing 80 the cellsuspension. While prior art freezing methods may be employed, the simpleplunge freezing method described herein for the freeze-thaw method mayalso be used for the freezing step 80 in the freeze drying protocol.

After freezing, a two stage drying process 150 is typically employed. Inthe first stage, energy of sublimation is added to vaporize frozenwater. When freeze drying biomaterials, the primary criterion forselecting the temperature of the primary drying phase is that it must bebelow the glass phase transition temperature of the freeze concentratedsolution to avoid collapse and undesirable chemical reactions. Ingeneral, the highest possible temperature that will not damage thesample should be used so that sublimation will occur quickly. Typically,the primary drying occurs at a constant temperature maintained below theglass transition temperature for the freeze concentrated solution.

Secondary drying is performed after the pure crystalline ice in thesample has been sublimated. Secondary drying cannot take place unlessthe temperature is raised above the glass phase transition temperatureof the freeze concentrated solute, however, it is crucial that sampletemperature not rise above the collapse temperature above which thespecimen is believed to mechanically collapse due to viscous flow.

Freeze dried materials can be stored 140, hydrated 160 and the cellporation reversed 170 in the same manner as described above for vacuumdrying. Viable cells may then be recovered 180.

EXAMPLE 1 FREEZE-THAW PROCEDURE

CELL CULTURE

Examples were performed using NIH/3T3 murine fibroblasts (American typeCulture Collection, Rockville, Md. cultured in Dulbecco's Modified EagleMedium (DMEM; Life Technologies, Inc., Gaithersberg, Md. supplementedwith 10% bovine calf serum (BCS, JRH Biosciences, Lenexa, Kans. andincubated at 37° C. with 10% CO₂ in air. At confluence, approximatelyevery three days, cells were trypsinized in 0.1% trypsin solution (LifeTechnologies) and replated at a 40 fold reduction in cell number.

CELL PORATION

Fibroblasts in the log-phase of growth were trypsinized in 0.1% trypsinsolution, washed twice in DMEM with BCS, and suspended (2×10⁶ cells/ml)in HEPES Buffered Saline (HBS:dH₂ O 130 mM NaC1, 7.2 mM KC1, 20 mM HEPES(N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]); all Sigma,St. Louis, Mo. at a pH of 7.4. As indicated in Table 1 and FIG. 3, HBSsuspending solution also contained either (i) 25 μg/ml of H5, thesite-directed zinc-switchable mutant of Staphylococcus aureus (S.aureus) α-toxin (produced and purified as in Walker et al., "Apore-forming protein with a metal-actuated switch," Protein Eng. 7,655-662 (1994)), (ii) 25 μg/ml of wild type S. aureus α-toxin (Sigma;WT), or (iii) no addition. Because of the variability among proteinpreparations, the hemolytic activities of the two porating proteins (H5and WT) were equalized by measuring the release of hemoglobin from raterythrocytes at OD 405 as adapted from Hildebrand et al.,"Staphylococcus aureus α-toxin: Dual mechanism of binding to targetcells," J. Biol. Chem. 266, 17195-17200 (1991). Though there was littledifference in their activity (50% release of hemoglobin at 0.1 μg/mlafter 3 hours of poration), the concentration of WT protein solution wasslightly reduced so that its activity matched the activity of the 25μg/ml of H5. Cells were incubated in the toxin containing solutions for10 min to allow for proper pore formation.

SUGAR LOADING

Following poration, 0-1.0 M trehalose (final concentration) in HBSsolution (Sigma) was added to the cell suspension, as indicated in FIG.4. To allow for trehalose uptake, porated cells were incubated in thesolution for 45 min. A 45 min interval has been demonstrated to besufficient for the uptake of sucrose, a disaccharide similar totrehalose. [Russo et al., "Reversible permeabilization of plasmamembranes with an engineered switchable pore," Nature Biotech. 15,278-282 (1997).] This uptake was measured by both the uptake ofradiolableled sugar into porated cells as well as by the volumetricresponse of porated cells placed in a hypertonic sugar solution,yielding statistically correlative results. By repeating the volumetricexperiments using hypertonic trehalose solutions and 25 μg/ml ofporating agent, volumetric equilibration over 90% over a 45 min intervalwas achieved, indicating, the equilibration of intracellular trehaloseconcentration with that of the suspending solution.

PRE-FREEZE EXPERIMENTS

As specified in Table 1 and FIG. 3, fibroblasts were treated withvarying poration and trehalose schemes by the methods described above.To determine the effects of these pre-freeze treatments on cellviability and function, cells were then assayed by flow cytometricanalysis and by measuring cellular plating efficiency, clone formingability, and population growth as described below.

CRYOPRESERVATION PROTOCOL

Following a variety of poration and trehalose treatments as indicated inFIGS. 3 and 4, 0.9 ml of the cell suspension (10⁶ cells/ml) was placedinto a 1.9 ml cryovial (Nalgene, Rochester, N.Y.), clipped into acryovial holder (TS Scientific, Perkaise, Pa.), and plunged into liquidnitrogen (LN₂) and stored there for 60 minutes. Individual vials werethen thawed in a 37° C. water bath with continuous, mild agitation for 5min. For later analysis by the methods described below, thawed cellswere diluted in serum containing DMEM which contained sufficient Zn²⁺ toseal the H5 pores and promote cell recovery.

FLOW CYTOMETRY

At 15 minutes, 1 hour, and 2 hours following either the pre-freeze cellpreparation (poration and trehalose loading) or after the freeze-thawprotocol, two fluorescent dyes, calcein (5 μM; Molecular Probes; DugeneOreg.) and ethidium homodimer (EH; 10 μM; Molecular Probes) were addedto HBS cell suspensions to assess viability of the cells. Calceinfluoresces green (510 nm) when retained intracellularly and indicates alive cell with an intact cell membrane, while EH fluoresces red (617 nm)when bound to DNA and thus stains the nucleus of a dead cell. Followingcalibration, cells were run at low flow rates through the BecktonDickinson FACSCalibur flow cytometer (San Jose, Calif.) to measureforward and side scatter as well as take emission readings at 530±30 nmand 63±022 nm of 10,000 particles. Cells, as indicated by size andganularity, were evaluated for their emissions at the two wavelengthsindicated above. High green and low red fluorescence was scored as live,while low green and high red were scored as dead; those displaying lowor high values for both were scored as dead. Since the live/dead data isdelivered as a percentage, total cell yield was determined by countingon a hemocytometer (Baxter, McGaw Park, Ill.). Those which do not appearin the total yield, presumably cells which have lysed and dispersed,less those lost by cell handling and transfer as determined by controlexperiments, were likewise scored as dead.

PLATING EFFICIENCY

Following either the pre-freeze cell preparation or after thefreeze-thaw protocol, each cell suspension was diluted in 12 ml DMEMwith BCS and plated in a P75 tissue culture flask (Falcon, FranlinLakes, N.J.). After three hours, flasks were washed twice with HBS andthose cells remaining in the flask were trypsinized and counted using ahemocytometer. For pre-freeze experiments, reported values are thenumber of cells which were successfully plated normalized to the totalnumber of cells input to the system following preliminary cell handling.For freeze-thaw conditions, reported values; are the number of cellswhich successfully plated normalized to the total number of cells inputto the system following preliminary cell handling and any toxintreatment. Micrograph images were obtained on a Nikon Diaphot microscope(Nikon Ph2, NA-.4; Tokyo, Japan) at 120x magnification and captured withMetamorph imaging software and are shown in FIGS. 2A-G.

CLONOGENIC ASSAY

For each condition, approximately 200 of the cells that had plated in 3hours were replated in a P75 for one week. To obtain 200 fibroblast,cells were diluted to 2×10⁵ cells/ml, and 1 μl of cell solution wasintroduced into a P60 petri dish (Falcon) containing 12 ml of DMEM withBCS. After one week, cells were fixed by a 0.5% glutaraldehyde wash andstained by Rhodamine B (Sigma) to enhance colony visibility. The numberof colonies formed was determined by visual inspection.

SURVIVAL AND GROWTH

Following either the pre-freeze cell preparation or after thefreeze-thaw protocol, the cell suspensions for each condition werediluted in 12 ral of DMEM with BCS and plated in P75 flasks. After 18hours, approximately two population doubling cycles, flasks were washedtwice with HBS. Fibroblast populations were then trypsinized and countedon a hemocytometer. For pre-freeze measurements, data was normalized tothe unporated control case with no trehalose addition. For post-thawexperiments, reported values are the total number of cells in each flasknormalized to an unfrozen control population otherwise treatedidentically to the experimental condition.

STATISTICAL ANALYSIS

Data were analyzed by ANOVA factorial and evaluated with Fisher'sPost-Hoc Test for at least 97% confidence (p≦0.03). Pre-freeze flowcytometry, plating efficiency, and clonogenic experiments were repeatedat least twice, and 18 hours growth experiments were repeated at leastthree times. For all experiments, at least two independent measurementswere performed. Data reported are normalized averages of total viableand functional cell populations at indicated time points±the standarderror of the mean (SEM).

EXAMPLE 2 VACUUM OR AIR DRYING PROCEDURE

The efficacy of intracellular trehalose for vacuum dried fibroblasts wasalso tested. NIH/3T3 murine fibroblasts, prepared as in Example 1, wereresuspended in phosphate buffered saline (PBS) solution and the celldensity was counted. Cell density was then adjusted to 100,000 cells/mlby diluting with PBS and the sample was divided into three equalaliquots of 4.0 ml each. To each aliquot, 1.0 ml of 25 μg/ml H5 wasadded, reducing cell density to 8×10⁴. The aliquots were incubated atroom temperature for 15 minutes with occasional mixing.

In the first two aliquots, enough 1.0M stock solution of eithertrehalose or sucrose in PBS was added to obtain a final sugarconcentration of 0.3M. In the third aliquot, an equivalent amount ofPBS, without sugar, was added.

The three aliquots were dispersed in triplicate wells at 20 μl/well intofour duplicate 12-well plates. Two of the twelve well plates wereincubated at 37° C. and 10% CO₂ as controls. The remaining two plateswere vacuum dried for 2 hours at ambient temperature. After drying, thedried samples were rehydrated and the poration reversed with serumcontaining DMEM.

Viability assays were performed by counting attached cells at 3 hours,and cells that remained attached and divided during the next 24 hours.The experiments were repeated three times. As shown in FIG. 5, anaverage of 48±3 cells that successfully attached at three hours for thetrehalose sample, and 36±3 for the sucrose sample. Adjusting for cellloss due to handling, poration, etc. using the control samples, survivalrates of 4% for the cells dried in trehalose and 3% for the cells driedin sucrose were obtained. Periodic microscopic examination of theattached cells over the next 24 hours indicated that the attached cellswere able to maintain the characteristic spread morphology and undergoproliferation.

RESULTS

The examples demonstrate that 0.2 M of intracellular trehalose alone issufficient to provide protection for over 80% of the 3T3 fibroblastscryopreserved in a simple liquid nitrogen plunge protocol. Thisconcentration is five-fold to an order of magnitude lower than thefunctional concentrations of traditional CPAs such as Me₂ SO andethylene glycol. Further, this protocol allowed for the preservation of1 ml samples, and did not have a CPA removal requirement. Based on theseresults, the method of the invention allows a simple cryopreservationprotocol for mammalian cells.

To illustrate this point, a variety of assays were implemented toevaluate the condition of the cells at various time points following theuptake of trehalose as well as after the freeze-thaw cycle. At earlytime points, 15 and 60 min after treatment, cell viability was evaluatedby assaying the integrity of the cell membrane with a double-labelfluorescent stain. Next, after three hours in culture, we tested thenormal ability of these cells to attach to the polystyrene substrate andspread, taking on an elongated spindle-like shape. Finally, cells whichattach should then secrete extracellular matrix and undertake thecomplex task of cell division. Because it involve, a complex series ofsteps involving DNA replication, spindle formation, and proteinsynthesis, mitosis is an ideal marker for the functional integrity offibroblasts. Thus, incorporating both the death of cells as well as theability of surviving cells to divide, the population's growth over 18hours is indicative of the overall freezing damage suffered by thecells.

As a baseline for the freezing results, the condition of cells followingporation and exposure to trehalose was evaluated. Overall, these datafor 18 hours growth indicate that over 95 % of fibroblasts can withstandthe osmotic shock of being placed into 0-0.4 M trehalose, with poratedcells suffering only an additional 10% loss. Since fibroblasts reachtheir minimum volume when exposed to about 0.4 M trehalose (data notshown), the excessive dehydration and osmotic forces caused byconcentrations higher than this threshold are most likely responsiblefor their loss of viability. Porated cells do not shrink to the samedegree as non-permeabilized cells due to the altered water and solutetransport properties of permeabilized cell membranes. They, therefore,seem to withstand the osmotic insult more readily. Moreover, theengineered H5 switch, in combination with this osmotic advantage, causesthe H5 treated cells to outperform both other conditions by 20-50% formuch of the upper range of trehalose concentrations. Based on these andprevious data, trehalose loading was targeted at 0-0.4 M for the designof freezing protocols. This range is comparable to the trehaloseconcentrations used for protein and virus preservation. Since trehaloseis non-toxic at these concentrations, freezing protocols were designedwithout a CPA removal step.

EFFECT OF PORATION AND TREHALOSE CONCENTRATION ON NON-FROZEN SAMPLES

To maximize the final post-thaw survival of the samples, the pre-freezetreatment steps were optimized, finding conditions for permeabilizationand trehalose loading which were minimally damaging to cells. Pre-freezecontrol experiments were performed under a variety of permeabilizationand trehalose conditions to measure the amount of cell loss attributableto each stage of treatment. Overall, large numbers of fibroblasts, over85%, maintained viability and function following permeabilization byeither WT or H5 either with or without trehalose addition. The completeset of pre-freeze control data is reported in Table 1. These data werenormalized to cell populations following a 4% loss resulting from cellhandling (pipetting, centrifugation, etc.) as measured by hemocytometer.

                  TABLE 1                                                         ______________________________________                                        Evaluation of Pre-freeze and Post Thaw Cells Porated and Loaded with           Trehalose                                                                                                           Survival                                                                        0.4M Flow Cytometry Plating                                                 Efficiency and Growth                    H5/WT Trehalose (1 h) (3 h) (18 h)                                          ______________________________________                                        Pre-Freeze Conditions                                                           --      -        94.5 ± 3.3                                                                           91.5 ± 1.5                                                                           100                                      -- +   98.4 ± 2.6                                                          WT - 95.0 ± 1.0 92.2 ± 1.8 90.0 ± 3.6                                WT + 78.0 ± 3.0 80.0 ± 3.0 87.4 ± 0.9                                H5 - 91.9 ± 4.6 87.5 ± 5.6 91.5 ± 3.9                                H5 + 85.1 ± 3.1  84.2 ± 5.8                                           Post-Thaw Conditions                                                            --      -         4.5 ± 3.5                                                                            0.0 ± 0.0                                                                            0.0 ± 0.0                            -- + 45.0 ± 8.8 11.2 ± 2.1 14.2 ± 8.9                                WT - 12.1 ± 4.9  0.0 ± 0.0  0.0 ± 0.0                                WT + 81.3 ± 1.2 19.3 ± 7.3 16.9 ± 3.6                                H5 - 13.2 ± 4.9  0.8 ± 0.8  0.0 ± 0.0                                H5 + 79.0 ± 6.4 66.1 ± 6.3 70.8 ± 2.1                              ______________________________________                                    

Specific experiments were performed to evaluate the effect of simplysuspending cells in HBS as would occur during the loading of trehalose.Flow cytometric analysis with live/dead stain indicated that 3.0±0.4% ofthe cell population was not viable following a two hour of exposure toHBS. At three hours, cells suspended in DMEM with serum plated andspread effectively with no additional losses. Finally, by 18 hours, thecell populations increased by a factor of 2.25, indicating a baselinefor normal cell growth and population doubling dynamics. Similarly, thesurvival and growth of non-porated cells suspended in 0.4M trehalose inHBS (98.4±2.6) was not statistically different from that of thepopulation suspended in HBS alone (p=0.777).

To better understand the consequences of the trehalose loading protocol,the effect of poration and trehalose treatment on the viability andfunction of unfrozen fibroblasts was quantified by evaluating thecondition of cells porated with 25 μg/ml of a porating agent, eitheralone or with 0.4 M trehalose by a battery of assays as shown inTable 1. Interestingly, none of the porated conditions was significantlydifferent from any other porated condition regardless of porating agent(i.e. WT vs. H5) and independent of the addition of trehalose (p<0.0300for all comparisons). Therefore, it appears that we can load 0.4 Mtrehalose into fibroblasts with minimal loss to the cell population. Inaddition, for all conditions tested, cells which were able to attach andspread at 3h were also able to form colonies after one week of culture.

In developing the freezing protocol, the effect of trehaloseconcentration on the survival of either unporated cells or cells poratedwith H5 or WT was measured. As reported in Table 2, cells exposed totrehalose did not demonstrate a significant loss of viability offunction for concentrations less than 0.4 M independent of poration(p>0.5000 for all comparisons). At 0.6 M, H5 porated cells demonstrated80% survival, thereby outperforming the WT and unporated conditions byover 20% (p=0.0010 and 0.0065, respectively). Finally, from 0.8 to 1M,H5 and WT treated populations suffer loss of half of their populations,while unporated cells retained only 10% viability. From this study, itappears that fibroblasts should preferably be exposed to less than orequal to about 0.4 M trehalose in order to maintain high viability.

                  TABLE 2                                                         ______________________________________                                        Survival of Pre-Freeze Cells: Dose Response of Trehalose                          Trehalose (M)                                                                            No Poration  WT     H5                                         ______________________________________                                        0.0        100          90.0 ± 3.6                                                                          91.5 ± 2.2                                  0.2 99.2 ± 3.3 95.6 ± 7.0 98.1 ± 6.4                                 0.4 98.5 ± 2.6 87.4 ± 0.9 84.1 ± 3.3                                 0.6 60.7 ± 3.3 52.3 ± 3.2 79.1 ± 3.0                                 0.8 39.4 ± 1.0 59.5 ± 4.4 52.0 ± 0.9                                 1.0  9.1 ± 1.6 44.5 ± 2.3 60.6 ± 2.6                               ______________________________________                                    

EFFECT OF PERMEABILIZATION AND INTRACELLULAR TREHALOSE ON PLUNGE-FROZENCELLS

The use of trehalose as an intracellular cryoprotectant was demonstratedby testing the survival and function of cells treated with 25 μg/ml ofH5 and 0.4 M trehalose, subsequently plunged in LN₂, and finally thawedin a 37° C. water bath. The results demonstrate the effectiveness oftrehalose as an intracellular CPA and illustrate the importance of theH5 switch. They are qualitatively outlined in the micrographs of FIG. 2.The first two panels (A, B) show unfrozen and untreated control samplesat 3 and 18 hours after plating. Large numbers of cells attach, spread,assume the spindle shape of fibroblasts, and subsequently divide. Thisis in sharp contrast to the frozen and untreated case (C, D) in whichall cells die as a result of the freeze-thaw process. Interestingly,though a moderate fraction of cells treated with WT and loaded withtrehalose appear to survive the freeze-thaw protocol (E), these cellsare neither able to sustain their viability nor divide properly,resulting in very small populations at 18 hours (F). On the other hand,H5 porated cells loaded with trehalose demonstrate a substantial amountof survival and high plating efficiency (G), as well as a normal degreeof cell division resulting in large populations at 18 hours (H).

These data, as well as cell viability at 1 hour, 3 hours and 18 hours,were quantified as shown in FIG. 3. Again, these data demonstrate theconsistently high viability and function of cells treated withintracellular trehalose loaded through H5 pores, as compared to allcontrol conditions. For the H5 and trehalose samples, at 1 hourpost-thaw, live/dead staining measured 79.0±6.4% viable cells. At threehours, 66.1±6.4% cells plated and spread with 211.6±5.2 colonies formedfrom approximately 200±12 of these functional cells. At 18 hours, thecells formed populations which were 70.8±2.1% of the size of controlpopulations which were likewise treated with H5 and trehalose but whichwere not frozen (Table 1). Moreover, these data show no significantdifference among time points, implying that cells which were alive at lhcontinued to be viable and functioning (p>0.1000 for all comparisons).In addition, these data are 70-80% higher than all control data to whichtrehalose was not added (p<0.0001). Further, though at 1 hour the H5 andWT (81.3±1.2%) conditions treated with trehalose are not statisticallydifferent (p=0.8073), the loss of over 60% of the WT condition'spopulation at 3h (19.3±7.3%) and 18 hours (16.9±3.6%), resulted in alarge and statistically significant difference between the use of WTand. H5 as the porating agent (p<0.0001). In fact, WT porated cellsloaded with trehalose performed so poorly, that their survival was notstatistically different from the case in which trehalose was simplyacting extracellularly (p>0.4000). Though non-frozen samples previouslyindicated little difference between WT and H5 treatment, these post-thawdata indicate a dramatic difference, implying that the engineered H5 isthe preferred porating agent for loading trehalose into cells prior thefreezing.

EFFECT OF TREHALOSE CONCENTRATION OF PLUNGE-FROZEN CELLS

Since the use of any intracellular CPA requires a balancing between theadded freezing protection afforded by higher concentrations and lossesdue to excessive osmotic forces and toxicity, the effect of trehaloseconcentration on H5 porated cell survival and growth at 18 hours wasmeasured. These data, as illustrated in FIG. 3, indicated an invertedU-shaped curve, with 0.2 M treated cells performing remarkably well withan 18 hour population of 79.2±4.0%. At 0.1 M the population wassignificantly lower at 28.6±3.4% (p<0.0001) while at higherconcentrations the population size steadily decreased, remainingstatistically significant from the 0.2 M condition (p<0.0010). As theconcentration increases, the curve flattens which leads to statisticallyinsignificant (p=0.9477) differences between 0.6 M (28.5±2.3%) and 0.8 M(29.4±2.1%). Overall, these data imply that for a freezing protocol witha one step trehalose loading process, 0.2 M is the preferredconcentration, providing over 80% survival and function with 3T3fibroblasts.

EFFICACY OF INTRACELLULAR SUGARS FOR VACUUM DRYING OF CELLS

Fibroblasts were also porated with H5 and loaded with 0.3 M trehalose orsucrose in order to test the efficacy of the preservation method fordried as well as frozen cells. One group of cells was porated with H5but loaded with PBS only with no sugar for use as a control. Adjustingfor cell loss due to handling, poration, etc. using the controls,survival rates of 4% for cells dried in trehalose and 3 % for cellsdried in sucrose were obtained using a simple ambient temperature vacuumdrying step. Periodic microscopic examination of the attached cells over24 hours indicated that the attached cells were able to maintain thecharacteristic speed morphology and undergo proliferation. Bothtrehalose and sucrose were thus shown to be effective in protectingcells subjected to vacuum drying.

CONCLUSIONS

The freezing experiments demonstrate that cells permeabilized with H5and loaded with less than or equal to about 0.4 M trehalose have adramatically higher level of viability and function as compared to allother tested conditions. In fact, all controls without trehaloseresulted in 100% mortality, while the H5 and trehalose combinationresulted in over 70% survival and growth. The presence of trehalose aswell as the reversible permeabilization afforded by H5 has multipleadvantageous effect, which may bolster cell survival. Primarily,intracellular trehalose, by increasing cytosolic viscosity, may inhibitthe growth of intracellular ice crystals, thus mitigating cell damage.Further, as has been demonstrated for isolated biomaterials, trehalosemay stabilize cell membranes and proteins during freezing, therebypreventing membrane leakiness and favoring native protein configuration.Likewise, sample stability may be improved by trehalose's ability topermit a higher temperature glass transition in the unfrozen fraction.

Though both the H5 and WT conditions have presumably similarintracellular trehalose contents, the H5 condition demonstrated 56%higher survival and growth. This result underscores the advantage of thezinc-actuated switch. Since DMEM with serum (post-thaw medium) containsμM concentrations of Zn²⁺ ions, the H5 pores are blocked under thesepost-thaw conditions. Thus, the cells treated with the engineered andswitchable H5 molecules and subsequently plated in DMEM with serumregain their natural permeability to small molecules, permitting areturn to normal homeostasis and subsequent recovery. Without thebenefit of the resealing, WT permeabilized cells cannot recover properlypost-thaw, and they ultimately die. As suggested above, under certainlimited circumstances it may be possible to reversibly permeabilizetarget cells with WT where the WT pores are unstable and are shed by thecells before the cells die off from the permeabilization effects.

Although the initial freezing experiments used 0.4 M trehalose, themaximum concentration indicated by pre-freeze evaluation, the effect ofvarying the trehalose concentration on the post-thaw survival ofH5-permeabilized cells was evaluated by varying the trehalose dose up to1.0 M. The resulting trehalose dose response is represented by aninverted U-shaped curve with a maximal survival of over 80%, at 0.2 M.Apparently, below 0.2 M there is not adequate trehalose present toafford proper protection. Above this, the loss of viability seems to bea direct result of exposing the cells to excessive osmotic pressureduring loading and of not removing the trehalose after thawing.

To probe the mechanism of trehalose protection and test whetherintracellular ice formation occurs under the exemplary conditions, thetemperature excursion of plunge-frozen H5 and trehalose treated sampleswas measured with a thermocouple, and the measured cooling ratesreproduced on a cryomicroscope. Fairly independent of position, plungedcells experienced a cooling rate of -70° C./min from 22° C. to -15° C.,and -160° C./min to -175° C. with a latent heat release at -12 to -15°C. When fibroblasts treated with H5 and loaded with trehalose weredeliberately exposed to this cooling protocol on a cryomicroscope, cellsdarkened following the seeding of extracellular ice, including thatintracellular ice had formed. If this indeed reflects what occurs in thecryovial, the trehalose must somehow protect the cell from the damagingeffects of intracellular ice formation.

Often, preservation protocols have been evaluated at times shortlyfollowing thaw, resulting in the presumption of success. The datapresented here demonstrates that such testing is not a reliableindicator of a protocol's effectiveness. For the plunge freezeexperiments, the earliest fluorescent tests for cell viability are notindicative of the final condition of cell populations. At 15 minutes,all frozen conditions, except untreated controls, demonstrate 60-80%viability. However, after one hour cells treated only with either H5 orWT retain 10% viability, and cells with trehalose added alone have 45%viability. Only cells treated with both a porating agent and trehaloseretain 70-80% viability and function after 1 hour. Further, as indicatedby plating at 3 hours and growth at 18 hours, the H5 and trehalosecondition leads to long term cell survival and function. These dataimply that damage due to freezing stresses, at least in part, does notresult in the immediate destruction of cells, but rather in a gradualloss of viability.

The Examples show the successful cryopreservation of 3T3 murinefibroblasts using less than or equal to 0.4 M trehalose as the sole CPAin a rather simple LN₂ plunge cryopreservation protocol. The trehaloseconcentration used is an order of magnitude lower than what is requiredfor mammalian cells in traditional constructs utilizing traditionalCPAs. Survival, however, is dependent on the reversible permeabilizationafforded by the H5 switchable pore. Further, freezing and thawing tookplace in a rather large sample volume (˜1 ml) and without any regard forthe cooling profile, seeding temperature, or plunge temperature. Thissuggests that the use of small carbohydrates introduced intracellularlycan allow the preservation of living material using low concentrationsof CPA, thus avoiding toxicity effects, and implementing simplifiedfreezing protocols.

The efficacy of intracellular trehalose was also shown for vacuum driedfibroblasts. Vacuum drying at ambient temperature of reversibly poratedfibroblasts loaded with trehalose or sucrose resulted in viable cellsafter rehydration.

It will be understood that the foregoing is only illustrative of theprinciples of the invention, and that various modifications can be madeby those skilled in the art without departing from the scope and spiritof the invention. All references cited herein are expressly incorporatedby reference in their entirety.

What is claimed is:
 1. A method for dry storing living nucleatedmammalian cells having cell membranes, comprising:a. Applying a membranetoxin to reversibly porate the cell membranes of the nucleated cells; b.Loading an agent having bio-preservation properties to a predeterminedintracellular concentration sufficient for preserving the cellularmaterial, the predetermined intracellular concentration being less thanor equal to about 1.0 M, the bio-preservation agent comprising a sugar;c. Drying the sugar loaded cells to a level sufficient to permit drystorage; d. Placing the dried cells in dry storage; e. Rehydrating thedried cells to a level sufficient for cell viability; and f. Reversingthe cell membrane poration to an extent sufficient to permit survivaland growth of the cells; wherein sugar is the only bio-protective agentemployed.
 2. The method of claim 1, wherein the cell membranes arereversibly porated using H5 α-toxin.
 3. The method of claim 1, whereinthe sugar having bio-preservation properties is selected from a groupconsisting of trehalose, sucrose, glucose and maltose.
 4. The method ofclaim 3, wherein the membrane toxin forms pores of at least about 2.0nanometers in the membrane.
 5. The method of claim 3, wherein thebiological material is loaded with an intracellular concentration ofsugar less than or equal to about 0.4 M.
 6. The method of claim 3,wherein the drying is accomplished by freeze drying.
 7. The method ofclaim 6, wherein the sugar loaded cells are plunge frozen to a cryogenictemperature.
 8. The method of claim 3, wherein the drying is a vacuum orair drying.
 9. The method of claim 8, wherein the drying is performed ata non-cryogenic temperature.
 10. A method for cryopreserved livingnucleated mammalian cells having cell membranes, consisting essentiallyof:a. Applying a membrane toxin to reversibly porate the cell membranesof the nucleated cells using H5 α-toxin; b. Loading the porated cellswith a bio-preservation agent consisting essentially of a sugar to apredetermined intracellular concentration sufficient for preserving thenucleated cells, the predetermined intracellular concentration beingless than or equal to about 1.0 M; c. Freezing the sugar loaded cells toa cryopreservation temperature; d. Storing the frozen biologicalmaterial at a cryo-storage temperature; e. Thawing the cryo-storedbiological material to a viable state; and f. Reversing the cellmembrane poration to an extent sufficient to permit survival and growthof the cells.
 11. The method of claim 10, wherein the sugar havingbio-preservation properties is selected from a group consisting oftrehalose, sucrose, glucose and maltose.
 12. The method of claim 11,wherein the intracellular concentration of the sugar is less than orequal to about 0.4 M.
 13. The method of claim 11, wherein the cells areplunge frozen.